Prrsv gp5 based compositions and methods

ABSTRACT

The disclosure includes compositions and methods for the production of an immune response against porcine reproductive and respiratory syndrome (PRRS) virus, or PRRSV. The disclosure is based in part on the use of two or more peptide domains, each with a different sequence, from the PRRSV GP5 protein ectodomain. Compositions and methods comprising polypeptides containing the two or more domains, or nucleic acids encoding them, are described.

RELATED APPLICATIONS

This application claims benefit of priority from U.S. Provisional PatentApplication 60/915,049, filed Apr. 30, 2007, and from U.S. patentapplication Ser. No. 12/111,871 filed Apr. 29, 2008, which are herebyincorporated in its entirety as if fully set forth.

FIELD OF THE DISCLOSURE

This disclosure includes compositions and methods directed to the use ofporcine reproductive and respiratory syndrome (PRRS) virus, or PRRSV,polypeptides in the generation of an immune response against thepolypeptide, and therefore PRRSV. The disclosure is based in part on therecognition that use of more than one GP5 ectodomain, differing insequence within an HV-2 hypervariable region, allows generation of abroader immune response against PRRSV than with the use of a singleectodomain. Also disclosed is the use of nucleic acid molecules encodingmore than one ectodomain to produce a broader immune response. Thedescription includes compositions containing polypeptides with more thanone of the ectodomains, or one or more nucleic acid molecule encodingthe polypeptides. Also described are methods to produce an immuneresponse by using polypeptides, nucleic acid molecules encoding them,and/or a composition of the disclosure.

BACKGROUND OF THE DISCLOSURE

PRRSV belongs to the family Ateriviridae, one of animal RNA virusfamilies Antigenic properties of PRRS viruses, like other RNA viruses,continually change, which results in a most problematic issue indeveloping an effective vaccine against this disease causing agent.However, there are fundamentals that are not changed in the PRRSVbiological system. Importantly, the virus infects a host cell of amulticellular organism for its replication or growth. To infect, thevirus must attach to a host cell as part of its life cycle. Forattachment, the virus must have a viral receptor recognition protein(RRP) that recognizes one or more specific receptors on the host cell.Last, the host cell's receptor generally does not change because it isusually required for a particular function and so not intended for virusrecognition.

But a virus utilizes the cell's receptor to attach or recognize the hostcell. Rather than modifying the receptor structure, an organismcontaining the host cell may produce antibodies that recognize the RRPof the virus to block attachment of virus to the host cell. Theantibodies are commonly referred to as neutralizing antibodies (NA). Inresponse, a population of virus often contains or produces modificationsto its RRP that allow escape from NA recognition. However, themodifications to the RRP are limited by the fact that the modified RRPmust still recognize the cellular receptor for virus attachment. If amodification results in a non-functional RRP, the virus cannot attach,and so cannot replicate or survive.

BRIEF SUMMARY OF THE DISCLOSURE

The disclosure relates to a three-dimensional arrangement of amino acidresidues present in the GP5 protein of porcine reproductive andrespiratory syndrome (PRRS) virus, or PRRSV. The arrangement ofresidues, or polypeptide domain, is present at the N-terminal portion ofthe GP5 protein, and has been referred to as the ectodomain of the GP5protein. The disclosure includes use of the domain in the context of apeptide, a polypeptide, a viral particle, or other protein containingcomposition. In some embodiments, the domain may be present in the formof a recombinant or fusion, peptide or polypeptide. In otherembodiments, the domain may be present in, or with, a recombinant viralparticle or virus. In further embodiments, a nucleic acid moleculeencoding a peptide or polypeptide containing the domain may be used toexpress the domain for the practice of the disclosure.

The disclosure is based in part on the unexpected discovery thatectodomains that vary in sequence within a previously unappreciated,HV-2 hypervariable region, may be selected for use in the preparationand use of materials to generate an immune response, including aprotective response, in an animal against PRRSV. In some cases, at leasttwo PRRSV isolates, each containing a different GP5 protein due to atleast one sequence difference within the HV-2 region, are prepared andused to generate an immune response. In other cases, at least three orfour PRRSV isolates, each containing a different GP5 protein due tosequence differences at least within the HV-2 region, are prepared andused to produce a protective response.

The disclosure includes the recognition that following a putative signalsequence, the GP5 ectodomain may be viewed as a combination of threeregions that precede a putative transmembrane region (or membranespanning domain or MSD). In sequential order from the N-terminus to theC-terminus of the GP5 protein, the regions are the HV-1 hypervariableregion (“HV1”), the conserved region (“CR”), and the HV-2 hypervariableregion (“HV2”), which is then followed by a putative transmembraneregion (“TR” or MSD). FIG. 1 provides a non-limiting example. Because ofthe identification of the HV2 as important to the generation of animmune response against PRRSV, the disclosure includes combinations ofat least two GP5 ectodomains where they differ in the sequence of theHV2. In some cases, the at least two ectodomains may be present in atleast two PRRSV isolates, which may be administered to produce an immuneresponse as described herein.

So in a first aspect, the disclosure includes a combination of at leasta first polypeptide domain and a second polypeptide domain, where eachdomain contains a conserved GP5 motif covalently linked to an HV2 andeach domain is antigenic in an animal subject to PRRSV infection. Inmany embodiments, the linkage is a peptide bond, or amide linkagebetween amino acid residues in a polypeptide. The GP5 motif and HV2 maybe contiguous such that the HV2 follows immediately after the motif inthe same polypeptide molecule. In other embodiments, the motif and HV2may be separated by a linker, such as one or more amino acid residues.In further embodiments, the motif and HV2 may be joined via a chemicallinkage other than a peptide bond.

This aspect of the disclosure includes alternative embodiments of thefirst and second polypeptide domains wherein at least one of the domainsis an expanded domain that further contains an HV1 covalently linked tothe conserved GP5 motif. This results in at least one domain containingat least three regions: the HV1, the conserved region (CR) containingthe conserved GP5 motif, and the HV2, in sequential order. Of courseembodiments of the disclosure include combinations of two, or more thantwo domains, such as three or four domains, where at least two of thedomains are expanded domains as described herein. In some cases, each ofthe domains in a combination is an expanded domain.

In some embodiments, a combination of at least two polypeptide domainsis a combination of at least two PRRSV isolates, each of which containsat least one of the domains. In many cases, each of the domains is anexpanded domain containing the HV1, the CR, and the HV2, where eachdomain is different because of at least one sequence difference withinthe HV2. Of course the domains may optionally contain other sequencedifferences, such as one or more differences in the HV1.

A combination of domains may be present in a combination of GP5polypeptides, each of which is present on a PRRSV isolate. Thus thedisclosure includes a combination of two or more isolates, eachcontaining a GP5 protein with an expanded polypeptide domain containinga different HV2 sequence as described herein. For example, and in anon-limiting combination of four isolates, a first polypeptide domain ispresent in a first isolate, a second polypeptide domain is present in asecond isolate, a third polypeptide domain is in a third isolate, and afourth polypeptide domain is present in a fourth isolate. Each of theisolates would differ from the others at least due to a different HV2sequence in a GP5 protein of the isolate. Of course other sequencedifferences, such as one or more differences in the HV1, may also bepresent in the isolates.

In polypeptide based embodiments beyond GP5 protein, the first andsecond polypeptide domains may be located on the same molecule or on twoseparate polypeptide molecules. The first and second domains eachcontain a conserved GP5 motif, represented by the amino acid sequenceC(E/S)LNG(T/A), SEQ ID NO:1. Embodiments of the disclosure includecombinations wherein the conserved GP5 motif in each of the two domainsis identical. Alternatively, the first and second domains may differ insequence, and so structure, via the limited variability (four possiblesequences) within the conserved motif as indicated by SEQ ID NO:1.

As described herein, each of the domains in a combination includes anHV2, the sequence of which differs among each of the domains. In casesof an expanded domain, the HV1 sequence may optionally also differbetween each of the domains. This is based in part upon the non-limitingview that an expanded domain containing the HV1, the CR, and the HV2forms a recognition “pocket” which should differ among the differentdomains of a combination to provide increased diversity when thecombination is used to produce an immune response. So by way of anon-limiting example, a sequence difference in the HV2 may result in analteration in “pocket” structure while sequence changes in both the HV2and the HV1 may result in a different alteration to the “pocket”structure. And while some embodiments of the disclosure include sequencechanges only in the HV2 and the HV1, other embodiments may includesequence changes in the CR.

In many embodiments, the HV2 contains about 8 amino acid residues and/ora conserved portion represented by the tripeptide sequence X₀WL, whereX₀ is one of the 20 naturally occurring amino acid residues. Thistripeptide sequence may be located at the beginning, or N-terminal end,of the HV2. In some embodiments, the conserved tripeptide sequencecomprises the sequence DWL, wherein X₀ is aspartic acid (D). In otherembodiments, X₀ is asparagine (N) or any other amino acid residue exceptaspartic acid (D). In further embodiments X₀ is an acidic amino acidresidue, such as glutamic acid (E) or glutamine (Q); a basic amino acidresidue, such as arginine (R), lysine (K), or histidine (H); an aminoacid residue with an aliphatic sidechain, such as alanine (A) orisoleucine (I) or glycine (G) or leucine (L) or valine (V); an aminoacid residue with a hydroxyl containing sidechain, such as threonine (T)or serine (S); or an amino acid residue with an aromatic sidechain, suchas tyrosine (Y).

In a second aspect, the disclosure is based upon the antigenicity and/orimmunogenicity of the conserved GP5 motif and HV2 in each of the domainsin a combination of two or more domains. In some embodiments, thepresence of a conserved GP5 motif and an HV2, and optionally an HV1 toform a recognition “pocket”, in each of at least two domains producesantigenic and/or immunogenic, and so the disclosure includes antigenicand immunogenic compositions containing the domains. In additionalembodiments, each of the domains is present in a separate polypeptidemolecule that is bound or associated with a cell membrane or other lipidbilayer. In some cases, the polypeptide molecule contains a TR, such asa GP5 transmembrane domain, which facilitates association with amembrane or lipid bilayer. In some embodiments, the membrane may be acell-free membrane or a fragment or portion of a cellular membrane, suchas an envelope or coat surrounding a viral particle produced by a cell.

Further compositions include two or more polypeptide molecules that aremembrane bound, or membrane associated, such as to a single viralparticle or to separate viral particles. The viral particle(s) may beinfectious or non-infectious, and independently, it may be replicationcompetent or incompetent. A viral particle may be a PRRSV particle orthat of another virus such as a recombinant viral particle that containsthe polypeptide molecules. Non-limiting examples of recombinant viralparticles that may be used to express a polypeptide of the disclosureinclude porcine adenovirus and poxvirus.

A viral particle that is both infectious and replication competent maybe referred to as a virion. So in some embodiments, the composition maycontain two or more polypeptide molecules that are membrane bound, ormembrane associated, such as to a single virion or to separate virions.In embodiments of compositions containing two or more virions, such astwo or more PRRSV particles, one or more may be a naturally occurringPRRSV particle or isolate that contains a first or second polypeptidedomain as described herein. In some cases, more than one, up to all, ofthe particles are naturally occurring isolates.

Of course additional embodiments of the disclosure include combinationsof more than two polypeptide domains or viral particles, each of whichcontains a conserved GP5 motif and an HV2, optionally with an HV1, asdescribed herein. So compositions comprising additional polypeptidedomains beyond a first and second polypeptide domain are expresslywithin the scope of the disclosure. Of course in such embodiments, theHV2, and optionally the HV1, in each polypeptide domain of a combinationdiffers in sequence, and so structure, from the HV2, and optionally theHV1, in each of the other domains in the combination.

In a further aspect, the disclosure includes a method of preparing acomposition described herein. In some embodiments, a method may compriseidentifying or selecting at least a first polypeptide domain and asecond polypeptide domain, each domain as described herein, andcombining the domains to form a composition. In many cases, a method maycomprise identifying or selecting at least a first polypeptide moleculecontaining the first domain and a second polypeptide molecule containingthe second domain, and combining the polypeptides to form a composition.In some cases, the combining may comprise addition of one or morepharmaceutically acceptable excipients and/or carriers in forming acomposition.

In other embodiments, the identifying or selecting may be among PRRSVisolates based upon the sequence of the HV2 in each isolate. In somecases, the identification or selection may be by detection of the HV2sequence along with one or more other portions of the GP5 molecule, suchas the conserved motif or the HV1. One non-limiting example of adetection method includes use of an antibody that recognizes a given HV2sequence, optionally in combination with, or in the context of, anotherportion of the GP5 molecule. Other non-limiting detection methodsinclude amino acid sequencing of the HV2 or nucleic acid sequencing ofthe sequence encoding the HV2.

In some embodiments, the detection may be of PRRSV in a sample of abiological fluid from an animal subject, such as an individual infectedwith PRRSV. The method may comprise contacting the sample, or a dilutedform thereof, with a binding agent which binds at least a portion of theHV2 in a GP5 protein. The sample may be from a porcine subject, but anysubject infected by PRRSV, or a PRRSV carrier, may be used. Thebiological fluid may be any fluid in which GP5 protein and/or PRRSVparticles may be detectably present. Non-limiting examples include thebodily secretions of a subject, such as saliva, tears, mucous, nasaldischarge, and vaginal secretions as well as other bodily fluids such asblood, serum, plasma, semen, seminal fluid, and urine as well as anyfluid component of feces or a fluid extract of feces.

In further embodiments, the identification, selection, or detection maybe of, or for, a novel PRRSV isolate that does not have an HV2 with asequence as disclosed herein. A novel isolate may be advantageously usedin a combination of the disclosure, such as with one, two, three, four,or more domain containing PRRSV isolates disclosed herein. A combinationwith a novel isolate would be expected to be advantageous because itwould have a higher likelihood of producing an antibody or immuneresponse which is novel when compared to the response to a combinationlacking the novel isolate.

As indicated above, an additional aspect of the disclosure is a methodof producing an antibody response (humoral immune response) or an immuneresponse. In some embodiments, a method may comprise administering acombination of polypeptide domains, as described herein, to an animalsubject with an immune system capable of producing the response. While agiven response may be viewed as including a response directed to thedomains or to polypeptides containing the domains, the disclosureincludes generation of a response that also recognizes GP5 in one ormore PRRSV isolates. In some embodiments, the antibody response includesthe production of one or more neutralizing antibodies. In otherembodiments, the immune response includes the production of one or morecellular immune responses, such as a T cell mediated response. In somecases, the antibody response or immune response is a protective responseagainst a PRRSV particle, such as one expressing a GP5 proteincontaining a polypeptide domain of the disclosure.

In some cases, the antibody response or immune response is against atleast two varieties, or strains, of PRRSV that differ in the HV2, suchas those likely to be present within a particular geographic region. Soembodiments of the disclosure include a response against one or morevarieties of a Lelystad isolate prevalent in Europe, one or morevarieties of a North American or Korean serotype of PRRSV, or one ormore varieties of PRRSV found in Asia or South America.

In additional embodiments, a method of producing an antibody or immuneresponse in a subject may comprise identifying or selecting, asdescribed herein, at least a first polypeptide domain and a secondpolypeptide domain, followed by administering the selected domains to asubject to produce the antibody or immune response. In some embodiments,the identifying or selecting may be of at least a first polypeptidecomprising the first polypeptide domain and a second polypeptidecomprising the second polypeptide domain, followed by administering theselected polypeptides to the subject. In many cases, at least one of thefirst and second polypeptides may be present in a PRRSV isolate. In somecases, each of the polypeptides is present in a PRRSV isolate.

In alternative embodiments, the identifying or selecting may be of atleast a first PRRSV isolate comprising the first polypeptide domain anda second PRRSV isolate comprising the second polypeptide domain,followed by administering the selected isolates to the subject. In someembodiments, the identifying or selecting is of at least three or atleast four, or more, isolates. In many cases, the selecting is basedupon the HV2 sequence in a GP5 protein of the PRRSV isolate. Theidentification or selection based upon the HV2 sequence may be performedby any suitable method, including, but not limited to, amino acidsequence analysis of the HV2, PCR-based or antibody-based detection ofthe HV2; or knowledge of the HV2 sequence in a previously characterizedPRRSV isolate.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages of the disclosure will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the amino acid sequences of arepresentative American type PRRSV strain (VR-2332) and a European typePRRSV strain (Lelystad strain, LV). The putative signal sequences ofeach are identified along with the HV1, “Conserved Region” or CR(containing the conserved GP5 motif), and the HV2 (underlined). Arepresentative, and non-limiting starting position for the HV1 is alsoindicated.

FIG. 2 is a schematic representation of groups of PRRSV isolates asdisclosed herein.

FIG. 3 shows an alignment of a portion of the GP5 ectodomain sequence,including the conserved GP5 motif and the HV2, from publicly accessiblePRRSV sequences. The NCBI (National Center for BiotechnologyInformation) accession numbers corresponding to the sequences areindicated along with isolates. The isolates include both North Americanand European strains as well as other types.

FIG. 4 illustrates GP5 protein mediated interactions between PRRSV and ahost pig cell.

FIG. 5 is a Kyte-Doolittle hydrophobicity plot of the amino acidsequence of GP5 protein. The indicated numbering is from an Americanisolate. There is a rapid shift to hydrophobic residues at about aminoacid residue 62, corresponding to the start of a putative transmembraneregion.

FIG. 6 provides the GenBank accession and GI numbers for representativePRRSV GP5 protein coding sequences.

DEFINITIONS

As used herein, the terms porcine reproductive and respiratory syndrome(PRRS) virus, or PRRSV, refer to a virus which has been reported tocause PRRS; Mystery Swine Disease (MSD); Swine Infertility andRespiratory Syndrome (SIRS), which was previously known as “blue-earedsyndrome”; porcine epidemic abortion and respiratory syndrome (PEARS);Wabash syndrome; mystery pig disease (MPD); swine plague; blue abortiondisease or blue ear disease in the United Kingdom; abortus blau in theNetherlands; seuchenhafter spatabort der schweine in Germany; andHeko-Heko disease (Shimizu et al., 1994). Additional alternative namesof the virally caused condition include Blue ear disease, Blue-eared pigdisease, Enfermedad misteriosa del cerdo, Epidemisch spätabort dersauen, Lane r bing (Chinese), Maladie blue du porc, Maladie mystérieusedu porc, Mystery pig disease, New pig disease, Plague of 1988-1989,Rätselhafte schweinekrankheit, Síndrome disgenésico y respiratorio delcerdo, Síndrome misterioso del cerdo, Syndrom reproductive etrespiratoire du porc, Syndrome dysgénésique et respiratoire du porc, andSyndrome HAAT (Hyperthermie-Anorexie-Avortement de la Truie).

The terms “GP5 protein” and “major envelope glycoprotein” of PRRSV asused herein refer to the polypeptide encoded by ORF5 of a PRRSV genomeas understood in the art. Representative, and non-limiting, GP5sequences coding sequences include those identified by the accession andGI numbers provided in FIG. 6. Without being bound by theory, andoffered to improve the understanding of the disclosure, GP5 proteinencoded by ORF5 of the PRRSV genome is believed to be a receptorrecognition protein (RRP) in PRRSV. The ectodomain of GP5 protein startsfrom about amino acid N30 to about D61 for the American strain and fromabout D33 to G63 for European strains (see FIG. 1). The typicaldifferences between American-type strains and European strains are (1)the total amino acids for GP5 proteins are 200 and 201, respectively,(2) European strains have a longer signal sequence compared to Americanstrains, and (3) European strains show less variations compared toAmerican strains. The disclosure is based in part upon the analysis, andidentification of the HV2 in each, of approximately 1740 GP5 sequencesand their respective ectodomains. Representative sequences are shown inFIG. 3. While the disclosure may be practiced with the use of thoserepresentative sequences, the disclosure is not limited to them.

The term “HV-1 region” or “HV1” refers to a polypeptide sequence presentat the N-terminal end of the conserved region of GP5 as describedherein. The region is optionally present in a polypeptide domain of thedisclosure. But when present, the sequence may be up to about 14 or moreamino acid residues in length, with lengths of 11, 12, 13 and 14 beingspecifically contemplated. In other embodiments, lengths of about 11,about 9, about 7, about 5, or about 3 or fewer residues may also beused. The disclosure includes embodiments wherein this region variesconsiderably in sequence. Non-limiting examples of HV1 sequences includethose present in FIG. 3.

Amino acid residues in the disclosed sequences may be conservativelysubstituted, or replaced, by another residue with similarcharacteristics and properties. As used herein, conservative amino acidsubstitutions of the disclosure are shown in Table 1 below.

TABLE 1 Definition Amino Acid Symbol Amino Acids with Aliphatic GlycineGly—G R-Groups Alanine Ala—A Valine Val—V Leucine Leu—L Isoleucine Ile—IAmino Acids with Hydroxyl Serine Ser—S R-Groups Threonine Thr—T AminoAcids with Sulfur- Cysteine Cys—C Containing R-Groups Methionine Met—MAcidic Amino Acids Aspartic Acid Asp—D Asparagine Asn—N Glutamic AcidGlu—E Glutamine Gln—Q Basic Amino Acids Arginine Arg—R Lysine Lys—KHistidine His—H Amino Acids with Aromatic Phenylalanine Phe—F RingsTyrosine Tyr—Y Tryptophan Trp—W Imino Acids Proline Pro—P

DETAILED DESCRIPTION OF MODES OF PRACTICING THE DISCLOSURE

General

The disclosure is based in part on an analysis of current PRRSV geneticinformation, such as the DNA sequences of the GP5 protein. Sequences ofPRRSV isolated from pigs showing clinical PRRS symptoms were alsoanalyzed. The analysis led to the identification of two hyper variableregions, HV-1 and HV-2, where the HV-2 region begins with either an X₀WLtripeptide motif wherein X₀ is one of the 20 naturally occurring aminoacid residues as described herein. The analysis also led to theidentification of a conserved region in positions I42 to T53 in anAmerican strain, and positions I44 to T55 in a European strain (see FIG.1).

Existence of a conserved region in the ectodomains among Americanstrains and European strains of PRRSV indicates that the conservedregion participates in direct contact between GP5 protein, as a receptorrecognition protein, and a receptor on a host cell to be infected byPRRSV. Based on this idea, and without being bound by theory, the two HVregions on either side of the conserved region are believed to serve as“gates” (or structural motifs) that maintain the hydrophobic propertiesof the conserved region. Previous commentaries on the HV-1 region andthe conserved region did not advance the studies of GP5 proteinimmunogenicity because there were too many variations in the HV-1 area.But in light of the hypervariability in HV-2, it was illogical to expectthat HV-2 would participate in interactions between GP5 and a host cellreceptor.

The instant disclosure is also based in part on the recognition thatvariations in the HV-1 region may be considered in combination with HV-2sequences that display less variation. Therefore, the instant disclosureincludes (1) sorting PRRSV isolates based upon HV2 sequence variationsto group them based upon immunological similarities; and (2) selectingcombinations of PRRSV strains in different groups to make broad spectrumvaccines that provide broader, heterologous protection uponadministration. The sorting and selection may optionally includeconsideration of the HV-1 region. Additionally, the isolates areoptionally first attenuated or inactivated prior to their administrationas a vaccine or immunogenic composition. Non-limiting examples ofattenuation include methods known to the skilled person, such as serialpassage in culture, such as in cells or tissue, or passage in animals.Of course the passaging may be conducted in vitro. Non-limiting examplesof inactivation include those known to the skilled person, such asheating, irradiation, chemical inactivation treatments.

The disclosure includes the optional division of all American strainsinto two groups based on amino acid position 61. More than 85% ofAmerican-type isolates have been reported to include D (Asp) or S (Ser)at position 61. The exceptions (less than 15%) usually have amino acidresidues at position 61 other than C (Cys), F (Phe), M (Met), W (Trp),and P (Pro). Therefore, the disclosure includes embodiments wherein thefirst HV2 residue, corresponding to position 61, is an amino acidresidue other than C, F, M, W, and P. In some embodiments, that residueis selected from A (Ala), G (Gly), V (Val), L (Leu), I (Ile), S (Ser), T(Thr), N (Asn), E (Glu), Q (Gln), R (Arg), K (Lys), H (His), or Y (Tyr).In alternative embodiments, however, that residue is selected from C, F,M, W, or P.

The disclosure includes the optional classification into two groups forAmerican strains; Group D and Group S, with eight sub-groups each (D-1through D-8 and S-1 through S-8, respectively) based on observedsequence information. The disclosure further includes division of allEuropean strains into eight (8) subgroups (E-1 through E-8) based onobserved sequence information. These groupings are illustrated in FIG.2.

The groups and subgroups are the basis for some embodiments of thedisclosure, where a combination of at least a first and secondpolypeptide domain (each containing a conserved GP5 motif covalentlylinked to an HV2 as described herein) from different groups orsubgroups, may be selected and used to produce a composition or vaccinethat produces a broader antibody or immune response than with use of thepolypeptide domains separately (or individually). In some embodiments, acombination of two to four, or more, polypeptide domains is used in thepractice of the disclosure. In further embodiments, the use of a domainfrom one group or subgroup may result in the production of an antibodyor immune response against more than one domain from the same group orsubgroup.

Non-limiting examples of the disclosure include combinations of at leastfour domains, wherein each of the four is selected, without duplication,from one of the 24 subgroups described herein as D-1 through D-8, S-1through S-8, and E-1 through E-8. A rough approximation of the number ofpossible combinations is provided by the mathematical formula(24×23×22×21)/(4×3×2×1), or about 10,600. But in some embodiments, thenumber of possible combinations are reduced significantly where eachcombination contains at least one domain from each of the Group D andGroup S subgroups as well as one from E-1 through E-8. A roughapproximation of such an example is provided by the formula(8×8×8×21)/(4×3×2×1), or about 448. In other embodiments where onlyGroup D and Group S subgroups are used, the number of possiblecombinations is also reduced. Similarly, embodiments where four domainsfrom E-1 through E-8 are used, the number of possible combinations arefurther reduced.

More generally, a composition or vaccine of the disclosure may includeat least one polypeptide domain from each of the D and S groups asdescribed herein. So a combination of two domains may have one from eachof the D and S groups. In other embodiments, a composition or vaccinemay include any combination of a D subgroup domain and/or anycombination of an S subgroup domain. So a combination of two domains mayhave both from Group D or both from Group S or one from each group. Inmany embodiments, the polypeptide domains used in a combination ispresent in the GP5 protein of a PRRSV isolate that is used as acomposition or vaccine of the disclosure. Therefore, the disclosure alsoincludes identification and classification of PRRSV isolates into thesame groups and subgroups described herein based upon the HV-2 sequencein the GP5 protein. The classified isolates may then be selected asdisclosed. As additional non-limiting examples, a composition or vaccineof the disclosure may contain

-   -   an isolate or domain from the D-1 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-2 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-3 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-4 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-5 subgroup and an at least one        or more (such as two or three or more) isolates or domains from        any other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-6 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup;    -   an isolate or domain from the D-7 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup; or    -   an isolate or domain from the D-8 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other D subgroup or any S or E subgroup.

Alternatively, a composition or vaccine of the disclosure may contain

-   -   an isolate or domain from the S-1 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-2 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-3 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-4 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-5 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-6 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup;    -   an isolate or domain from the S-7 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup; or    -   an isolate or domain from the S-8 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other S subgroup or any D or E subgroup.

Similarly, embodiments of the disclosure include a composition orvaccine of the disclosure may contain

-   -   an isolate or domain from the E-1 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-2 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-3 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-4 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-5 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-6 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup;    -   an isolate or domain from the E-7 subgroup and at least or more        (such as two or three or more) isolates or domains from any        other E, D, or S subgroup; or    -   an isolate or domain from the E-8 subgroup and at least one or        more (such as two or three or more) isolates or domains from any        other E, D, or S subgroup.

In some embodiments, however, a combination of the disclosure is not acombination of the GP5 ectodomains of VR2332 and LV as described herein.In other embodiments, a combination of the disclosure is not acombination of only GP5 ectodomains with the following sequencesbridging the boundary between HV2 and the putative transmembrane regionof GP5:

(SEQ ID NO: 7) ANKFDWAVET (SEQ ID NO: 8) ANKFDWAVEP (SEQ ID NO: 9)AGEFDWAVET (SEQ ID NO: 10) ADKFDWAVEP (SEQ ID NO: 11) ADRFDWAVEP or(SEQ ID NO: 12) SSHFGWAVET.

But specifically contemplated embodiments of the invention includecombinations of domains wherein both the X₀ residue in X₀WL and at leastone additional residue in the HV2 sequence both differ between thedomains of a combination.

As described herein, each polypeptide domain (and so each isolate)contains the conserved GP5 motif represented by the amino acid sequenceC(E/S)LNG(T/A), SEQ ID NO:1. So embodiments of the disclosure includedomains wherein the GP5 motif is represented by the amino acid sequenceCELNGT (SEQ ID NO:2), CELNGA (SEQ ID NO:3), CSLNGT (SEQ ID NO:4), orCSLNGA (SEQ ID NO:5). In other embodiments, the conserved GP5 motif islarger and is represented by the amino acid sequenceI(Y/F)(N/D/S/K)(L/S/F/M)(T/P/M)(L/I)C(E/S)LNG(T/A), SEQ ID NO:6, whichcorresponds to the “Conserved Region” shown in FIG. 1.

Virus Based Compositions

The disclosure is based upon the antigenicity and/or immunogenicity ofthe conserved GP5 motif and HV2 in a polypeptide domain, used incombination, as described herein. Thus the disclosure includescombinations of viral isolates as described above. Non-limiting examplesinclude combinations of the viral isolates listed in FIG. 3 (based ontheir sequence deposit information) as they may be classified into thegroups and subgroups disclosed herein. In some embodiments, combinationsof at least two or more, such as three or four or more, of thoseisolates are contemplated for use in the practice of the disclosure.

Additionally, the disclosure includes a combination of a viral isolateand a virus particle where each contains a polypeptide of a combinationdisclosed herein. In other embodiments, two or more virus particles areused. Non-limiting examples of a polypeptide domain containing virusparticle of the disclosure include an infectious or non-infectious virusparticle, which is independently replication competent or incompetent.Additional non-limiting examples include a virus particle cultured orpassaged in vitro; an attenuated virus; and a recombinant viralparticle.

In many embodiments, a viral particle is a PRRSV particle with an outermembrane that contains a GP5 protein with a polypeptide domain of thedisclosure. In other embodiments, the viral particle may be non-PRRSvirus with an outer membrane containing a polypeptide domain, optionallyas part of a GP5 protein, as described herein. Further embodimentsinclude a PRRSV or non-PRRSV viral particle with an outer membranecontaining two or more of the disclosed polypeptide domains, such as viatwo or more GP5 proteins with different ectodomains as described herein.In some cases, a viral particle is a PRRSV with a genome that containsmultiple copies of GP5 protein encoding ORF5 sequences. Such virusisolates have been previously reported and referred to as an“overproduction mutant” or “high-replication phenotype” PRRSV. Theinstant disclosure includes such a PRRSV that has been recombinantlymodified to contain and express more than one GP5 protein, eachcontaining a polypeptide domain with a different HV2 region as describedherein. In other embodiments, a recombinant virus may be an insectvirus, such as Baculovirus, which has been previously reported ascapable of expressing PRRSV GP5 protein, a porcine adenovirus, or apoxvirus.

In further embodiments, a virus isolate or viral particle is one that isinfectious and replication competent, such as a PRRSV isolate orinfectious particle. In most cases, the particle contains a genomeencoding and capable of expressing GP5 protein after infection in vivoto produce GP5. A particle that is both infectious and replicationcompetent may be referred to as a virion. In alternative embodiments, aparticle of the disclosure is infectious and replication incompetent,but optionally capable of intracellularly expressing GP5 proteins.

In embodiments comprising the use of a PRRSV isolate, the isolate may beidentified or selected based upon the sequence of the HV2 in an isolate.In some cases, such as that of an isolate represented in FIG. 3 herein,the identification or selection may be based upon review of the sequenceinformation or based upon knowledge of the HV2 sequence in acharacterized isolate. In other cases, such as where the isolate has notbeen previously characterized, the selection may be by detection of theHV2 sequence, such as by use of an antibody that recognizes a given HV2sequence; sequencing the GP5 coding sequence (ORF5) of the isolate; orpurification and amino acid sequencing of the GP5 protein per se.Non-limiting examples of antibody based detection includeimmunoprecipitation and assays such as ELISA, RIA, and Western blotting.Non-limiting examples of sequencing include dideoxynucleotide-basedsequencing of DNA molecules and PCR-based sequencing, including methodsbased upon reverse transcription of a GP5 encoding RNA molecule followedby PCR. In some embodiments, the selection of an isolate includesdetection of the sequence of one or more portions of the GP5 proteinbeyond the HV2, such as the conserved motif and/or the HV1.

In many embodiments of an antibody based detection method, the antibodydoes not bind to the GP5 protein as found in multiple PRRSV strains andisolates. Instead, the antibody should be sufficiently specific to theHV2 such that it is capable of detecting a particular isolate based inwhole or in part on the HV2 sequence or structure. In addition to theuse of an antibody, such as a labeled antibody to facilitate itsdetection, an antibody fragment that binds the HV2 of a PRRSV GP5protein may also be used. The antibody fragment may be the Fv or Fabregion of an HV2 binding antibody; other non-limiting examples include asingle chain antibody, including a single chain Fv region and a singlechain Fab region. The antibodies and antibody fragments are preferablymonoclonal but may be polyclonal in some cases.

In further embodiments, the detection of a PRRSV isolate is by use of asample of a biological fluid from a porcine subject, such as anindividual infected with PRRSV. The method may comprise contacting thesample, or a diluted form thereof, with a binding agent which binds theHV2 of GP5 protein, preferably to the exclusion of other moleculespresent in the biological fluid. In many embodiments, the subject is apig, and the sample may be of a bodily fluid or secretion from a pig.Non-limiting examples of pigs that from which samples may be obtainedfor use with the present disclosure include boar, sow, fattener, andgilt. The pigs may range in age from 1 to about 30, 31 to about 40, 41to about 50, or 51 to about 60 days or older.

Of course the biological fluid should be a fluid in which GP5 proteinand/or PRRSV particles are detectably present. Non-limiting examplesinclude bodily secretions such as saliva, tears, mucous, nasaldischarge, and vaginal secretions as well as other bodily fluids such asblood, serum, plasma, semen, seminal fluid, and urine as well as anyfluid component of feces or a fluid extract of feces.

Where the biological fluid contains PRRSV particles, detection may be byuse of a PCR-based method to detect a GP5 protein encoding nucleic acidmolecule, such as a DNA or RNA molecule containing a GP5 protein or aportion of the molecule encoding at least the HV2.

In additional embodiments, the selection and detection may be of, orfor, a PRRSV isolate that has a GP5 protein with an HV2 sequence thatdiffers from any disclosed herein or as previously characterized. Such anovel isolate may still be classifiable into one of the groups orsubgroups as disclosed herein. Alternatively, such an isolate may not beclassifiable into one of the disclosed groups or subgroups and so may beadvantageously used as part of a disclosed combination because the novelisolate would have a higher likelihood of producing a novel antibody orimmune response.

The disclosure thus includes a method of producing an antibody or immuneresponse in a subject by use of a PRRSV isolate comprising a GP5 proteinwith an HV2 sequence that differs from any HV2 sequence disclosedherein. The HV2 of the isolate may thus not be any described herein orencompassed by any of the disclosed groups or subgroups. The method maycomprise identifying a PRRSV isolate as comprising a GP5 polypeptidemolecule containing an HV2 region distinct from any HV2 sequence of FIG.3, or any D, S, or E subgroup, and administering said isolate to saidsubject to produce an antibody or immune response in said subject. Theidentifying or determining of a distinct HV2 sequence may be by anymeans disclosed herein, including an antibody or nucleic acid basedmethod as non-limiting examples, followed by comparison to the instantdisclosure. In some embodiments, the isolate is attenuated orinactivated as described herein.

Polypeptides and Compositions

The disclosure is based upon the antigenicity and/or immunogenicity of apolypeptide domain containing the conserved GP5 motif and HV2 asdescribed herein. The HV2 portion contributes to the antigenicity and/orimmunogenicity of the domain such that the use, in combination, ofpolypeptide molecules containing two different domains, results in thegeneration of a broader antibody or immune response in comparison to useof only one of the domains. Accordingly, the disclosure includescombinations of two or more polypeptide domains, such as in acomposition or vaccine, as well as their use in a method of immunizing asubject.

The nature of a polypeptide domain has been described herein. Generally,the domain contains a conserved GP5 motif covalently linked to an HV2region. Many embodiments have a peptide bond, or amide linkage, linkingthe GP5 motif and the HV2 so that they are contiguous when consideringthe sequence from N-terminus to the C-terminus. Other embodimentsinclude the use of a linker moiety. Non-limiting examples of a linkermoiety include a short peptide sequence, such as about 1, 2, 3, 4, or 5amino acids in length, and a non-peptide linker, such as a short chainof atoms with at least one carbon atom in the chain or other syntheticlinker. In cases of a short peptide sequence, the amino acids may be anynaturally occurring amino acid, such as the 20 amino acids of Table 1herein. In some alternative embodiments, the motif and HV2 may becovalently joined via a non-peptide bond linkage, such as acarbon-carbon bond.

With the use of first and second polypeptide domains, the domains may belocated on the same polypeptide molecule or two separate molecules. Inmany embodiments, the domains are located on separate polypeptidemolecules, each of which includes a transmembrane domain or otherprotein domain that allows for association with a lipid bilayer. Atransmembrane domain may also be present in a single polypeptidemolecule contain both domains. In some embodiments, the transmembranedomain is the putative transmembrane region of a PRRSV GP5 protein asknown to the skilled person and as described herein.

In many embodiments, the domains are located on separate GP5 proteins.In numerous other embodiments, the domains have identical sequences inthe conserved GP5 motif, such as that represented by CELNGT (SEQ IDNO:2). But even with an identical conserved GP5 motif, the first andsecond polypeptide domains differ in the HV2 sequence, which accountsfor the desired difference in antigenicity and/or immunogenicity betweenthe domains.

In many embodiments of separate GP5 proteins containing the first andsecond polypeptide domains, each GP5 protein may comprise, from theN-terminus to the C-terminus, a putative signal sequence, an HV-1hypervariable region, a conserved region (CR) containing the conservedGP5 motif, the HV-2 hypervariable region, a putative transmembraneregion, and the remainder of the GP5 protein. In other embodiments, aGP5 protein may lack all or part of the putative signal sequence. Ofcourse polypeptide molecules retaining the antigenic and/or immunogenicproperties of the disclosed polypeptide domains, but with fewer GP5components, may also be used. Non-limiting examples include apolypeptide molecule comprising the HV-1 hypervariable region, aconserved region (CR) containing the conserved GP5 motif, and the HV-2hypervariable region, optionally with a transmembrane domain asdescribed above.

Generally, a disclosed HV2 region is about 8 amino acid residues inlength. In alternative embodiments, the length may be 6, 7, 8, 9, or 10residues in length. The exact number of residues is unimportant so longas the resultant domain retains the desired antigenic and/or immunogenicactivity. In some embodiments, the HV2 begins with the tripeptidesequence X₀WL where X₀ is as defined herein. So in some embodiments, theHV2 is represented by the sequence X₀WLX₁X₂X₃X₄X₅, wherein each of X₀,X₁, X₂, X₃, and X₄ is independently one of the 20 naturally occurringamino acid residues shown in Table 1, and X₅ is selected from any aminoacid residue, with optional exception of C (Cys), F (Phe), M (Met), W(Trp), and P (Pro).

In other embodiments, the HV2 is a D group sequence represented byX₀WLX₁X₂X₃X₄D, wherein the aspartic acid (D) residue (at the end ofX₀WLX₁X₂X₃X₄D) may be replaced by any amino acid residue except C, F, M,P, W, S, T, and Y (such as replacement by A, G, V, L, I, N, E, Q, R, K,or H) and where X₀ is as described above, and one of subgroups D-1through D-8, which are represented by the following

D-1: wherein X₁ is an aliphatic amino acid residue, X₂ is an acidicamino acid residue (or wherein X₁ is an acidic amino acid residue and X₂is an aliphatic amino acid residue), X₃ is a basic amino acid residue,and X₄ is an amino acid residue comprising an aromatic ring, such asphenylalanine (F);

D-2: wherein X₁ is an aliphatic amino acid residue, X₂ is Ser, Thr, Tyror an basic amino acid residue (or wherein X₁ is Ser, Thr, Tyr or abasic amino acid residue and X₂ is an aliphatic amino acid residue), X₃is a basic amino acid residue, and X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F);

D-3: wherein each of X₁ and X₂ is independently an aliphatic amino acidresidue, X₃ is a basic amino acid residue, and X₄ is an amino acidresidue comprising an aromatic ring, such as phenylalanine (F);

D-4: wherein X₁ is an acidic amino acid residue, X₂ is Ser, Thr, Tyr ora basic amino acid residue (or wherein X₁ is Ser, Thr, Tyr or a basicamino acid residue and X₂ is an acidic amino acid residue), X₃ is abasic amino acid residue, and X₄ is an amino acid residue comprising anaromatic ring, such as phenylalanine (F);

D-5: wherein each of X₁ and X₂ is independently an acidic amino acidresidue, X₃ is a basic amino acid residue, and X₄ is an amino acidresidue comprising an aromatic ring, such as phenylalanine (F);

D-6: wherein each of X₁ and X₂ is independently one of the 20 naturallyoccurring amino acid residues, X₃ is an acidic amino acid residue, andX₄ is an amino acid residue comprising an aromatic ring, such asphenylalanine (F);

D-7: wherein each of X₁ and X₂ is independently one of the 20 naturallyoccurring amino acid residues, X₃ is a non-aromatic amino acid residuewith a hydroxyl containing R-group (such as Ser or Thr), and X₄ is anamino acid residue comprising an aromatic ring, such as phenylalanine(F); or

D-8: wherein each of X₁ and X₂ is independently either a basic aminoacid residue or an amino acid residue comprising an aromatic ring, suchas tyrosine (Y), serine (S), threonine (T), or phenylalanine (F), X₃ isa basic amino acid residue, and X₄ is an amino acid residue comprisingan aromatic ring, such as phenylalanine (F).

In additional embodiments, the HV2 is an S group sequence represented byX₀WLX₁X₂X₃X₄X₅ (where X₀ is as described above) and one of subgroups S-1through S-8, which are represented by the following

S-1: wherein X₁ is an acidic amino acid residue, X₂ is asparagine (N),X₃ is a basic amino acid residue, X₄ is an amino acid residue comprisingan aromatic ring, such as phenylalanine (F), and X₅ is S;

S-2: wherein each of X₁ and X₂ is independently an acidic amino acidresidue except that X₂ is not asparagine (N), X₃ is a basic amino acidresidue, X₄ is an amino acid residue comprising an aromatic ring, suchas phenylalanine (F), and X₅ is S;

S-3: wherein X₁ is an aliphatic amino acid residue, X₂ is an acidicamino acid residue (or wherein X₁ is an acidic amino acid residue and X₂is an aliphatic amino acid residue), X₃ is a basic amino acid residue,X₄ is an amino acid residue comprising an aromatic ring, such asphenylalanine (F), and X₅ is S;

S-4: wherein X₁ is an aliphatic amino acid residue, X₂ is Ser, Thr, Tyror a basic amino acid residue (or wherein X₁ is Ser, Thr, Tyr or a basicamino acid residue and X₂ is an aliphatic amino acid residue; or whereeach of X₁ and X₂ is independently Ser, Thr, Tyr or a basic amino acidresidue; or where each of X₁ and X₂ is independently an aliphatic aminoacid residue), X₃ is a basic amino acid residue, X₄ is an amino acidresidue comprising an aromatic ring, such as phenylalanine (F), and X₅is S;

S-5: wherein X₁ is an acidic amino acid residue, X₂ is Ser, Thr, Tyr ora basic amino acid residue (or wherein X₁ Ser, Thr, Tyr or is a basicamino acid residue and X₂ is an acidic amino acid residue except N(Asn)), X₃ is a basic amino acid residue, X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F), and X₅ is S;

S-6: wherein X₁ is a basic amino acid residue, X₂ is an asparagine (N),X₃ is a basic amino acid residue, X₄ is an amino acid residue comprisingan aromatic ring, such as phenylalanine (F), and X₅ is S;

S-7: wherein each of X₁ and X₂ is independently one of the 20 naturallyoccurring amino acid residues, X₃ is a basic amino acid residue, X₄ isan amino acid residue comprising an aromatic ring, such as phenylalanine(F), and X₅ is T or Y; or

S-8: wherein X₁ is an acidic amino acid residue, X₂ is an acidic aminoacid residue (or wherein X₁ is an acidic amino acid residue and X₂ is analiphatic amino acid residue, or alternatively wherein X₁ is analiphatic amino acid residue and X₂ is an acidic amino acid residue), X₃is an acidic amino acid residue, X₄ is an amino acid residue comprisingan aromatic ring, such as phenylalanine (F), and X₅ is S.

In yet additional embodiments, the HV2 is an E group sequencerepresented by one of subgroups E-1 through E-8 as follows:

the sequence NWLSX₂X₃X₄X₅ (represented by E-1), wherein each of X₂, X₃,and X₄ is independently one of the 20 naturally occurring amino acids,and X₅ is an acidic or aliphatic amino acid residue;

the sequence X₀WLX₁X₂X₃X₄X₅ (represented by E-2), wherein X₀ is anacidic amino acid residue except for asparagine (N), each of X₁, X₂, X₃,and X₄ is independently one of the 20 naturally occurring amino acidresidues, and X₅ is an acidic or aliphatic amino acid residue;

the sequence X₀WLX₁X₂X₃X₄X₅ (represented by E-3), wherein X₀ is a basicamino acid residue, each of X₁, X₂, X₃, and X₄ is independently one ofthe 20 naturally occurring amino acid residues, and X₅ is an acidic oraliphatic amino acid residue;

the sequence X₀WLX₁X₂X₃X₄X₅ (represented by E-4), wherein X₀ is anynon-acidic and non-basic amino acid residue, each of X₁, X₂, X₃, and X₄is independently one of the 20 naturally occurring amino acid residues,and X₅ is an acidic or aliphatic amino acid residue;

the sequence NWLSX₂X₃X₄X₅ (represented by E-5), wherein each of X₂, X₃and X₄ is independently one of the 20 naturally occurring amino acidresidues, and X₅ is Serine (S) or Threonine (T);

the sequence X₀WLX₁NX₃X₄X₅ (represented by E-6), wherein X₀ is any aminoacid residue except asparagine (N), each of X₁, X₃, and X₄ isindependently one of the 20 naturally occurring amino acid residues, andX₅ is Serine (S) or Threonine (T);

the sequence X₀WLX₁X₂X₃X₄X₅ (represented by E-7), wherein X₀ is anacidic amino acid residue except asparagine (N), each of X₁, X₂, X₃, andX₄ is independently one of the 20 naturally occurring amino acidresidues except that X₂ is not asparagine (N), and X₅ is any non-acidicamino acid residue; or

the sequence X₀WLX₁X₂X₃X₄X₅ (represented by E-8), wherein X₀ is anynon-acid amino acid residue, each of X₁, X₂, X₃, and X₄ is independentlyone of the 20 naturally occurring amino acid residues, and X₅ is anynon-acidic amino acid residue.

The term “non-acidic” refers to an amino acid residue other than anacidic amino acid; and the term “non-basic” refers to an amino acidresidue other than a basic amino acid.

In embodiments of separate polypeptide molecules containing the firstand second polypeptide domains, the polypeptide molecules may beadministered together or separately in the methods disclosed herein.When administered together, they may be formulated as a composition.Optionally, the composition comprises one or more acceptable carriers orexcipients or adjuvants as desired by the skilled person.

Methods of Preparation

The disclosure includes a method of preparing polypeptide domains andpolypeptide molecules as described herein. In some embodiments, apeptide or short polypeptide may be prepared by use of de novosynthesis, such as by automated chemical methods known to the skilledperson. Alternatively, the preparation may be by use of recombinant DNAmethods based upon the availability of nucleic acid molecules encodingthe polypeptide domains and polypeptide molecules of the disclosure. Thesequences of the nucleic acid molecules may be modified by knowntechniques, such as, but not limited to, PCR-based mutagenesis and denovo synthesis of nucleic acid molecules, such as by automated chemicalmethods known to the skilled person.

A method based upon the use of recombinant DNA techniques may be used toproduce a disclosed polypeptide. Such a method may comprise expressing anucleic acid molecule in a suitable expression system, such as an invitro cell culture system or in a producer animal, and isolating theexpressed polypeptide from the expression system. The expression systemmay comprise a nucleic acid sequence encoding a disclosed polypeptideand operably linked to a suitable regulatory or promoter sequence.Non-limiting examples of a suitable cell or cell line include porcinealveolar macrophages, CRL 11171, MA-104, MARC-145, PSP-36, andPSP-36-SAH. A non-limiting example of a producer animal is a pig, suchas a boar, sow, fattener, or gilt.

After producing a disclosed polypeptide domain, the method may compriseselecting and/or combining it as a first polypeptide domain with asecond polypeptide domain as described herein to form a composition. Thecombining may comprise adding one or more acceptable carriers,excipients and/or adjuvants to form a composition.

In some embodiments, such as with a PRRSV based nucleic acid molecule,the expression system produces viral particles that incorporate adisclosed polypeptide within the particle's outer membrane. The PRRSVbased nucleic acid molecule may be a viral genome that has been modifiedto express a GP5 protein containing an HV2 region as disclosed herein.In further embodiments, the nucleic acid molecule contains more than onecopy of a GP5 protein encoding sequence, where each copy encodes adifferent HV2 region as described herein. In other embodiments, theexpression system is cell-free, such as in the case of a rabbitreticulocyte system.

Other methods of producing PRRSV particles are also provided. In someembodiments, the production comprises selection and/or isolation ofPRRSV isolates as described herein. The selection and/or isolation maycomprise culturing or passaging an isolate as known to the skilledperson or as described herein. In alternative embodiments, the selectionmay be of an isolate from an infected subject, such as a pig, andfurther comprise obtaining infectious fluid and/or tissue from thesubject for use as a source of an HV2 region as described herein.Non-limiting examples of an infectious fluid and/or tissue includeblood, serum, plasma, nasal secretion, semen, seminal fluid, and urineas well as lung tissue, tonsil tissue, lymph node tissue, a fluidcomponent of feces or a fluid extract of feces. In some embodiments, theinfectious fluid and/or tissue may be used as part of a disclosedcombination. Non-limiting examples include use of a fluid or tissue asan inoculum in combination with a second HV2 region, optionally in apolypeptide molecule or a viral particle as described herein.

Methods of Use

The disclosure includes a method of generating an antibody or immuneresponse in a subject via administration of a disclosed combination offirst and second polypeptide domains. In some embodiments, the methodcomprises administration of a disclosed composition in an amounteffective to produce an antibody and/or immune response. In many cases,the administered amount is effective to produce a protected state in atreated subject against a subsequent challenge by one or more PRRSVisolates, such as infection by PRRSV. In some cases, a method mayfurther comprise an additional administration of a disclosed compositionas a “booster”. Non-limiting examples of the subject include a sow, agilt, a pregnant sow, or a pregnant gilt. In some embodiments, thesubject is a pig from about 1 to 12 weeks in age, such as about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, orabout 11 weeks. In other embodiments, the pig is from about 12 to about56 weeks or older in age, such as about 14, about 16, about 18, about20, about 22, about 24, about 26, about 28, about 30, about 32, about34, about 36, about 38, about 40, about 42, about 44, about 46, about48, about 50, about 52, or about 54 weeks. In additional embodiments,the pig has been weaned and/or has passed the stage at which maternalantibodies provide adequate protection.

An effective amount of a disclosed combination or composition, as avaccine, to produce a protected state in a subject, such as a pig, mayalso be determined by administration of the vaccine to an unaffectedpig, followed by challenge with PRRSV isolate. In some cases, theisolate may be purified or isolated in that its virus particles have thesame genome or the same GP5 protein or the same GP5 ectodomain.Non-limiting examples of isolates for use in a challenge include thoselisted in FIG. 3 as well as an infectious bodily fluid or tissue from ananimal infected with the isolate. In some embodiments, the challenge maybe after about 3 to about 8 weeks after a booster vaccination, and maybe with a large or excess amount of PRRSV.

The vaccine or amount thereof is effective if it reduces the severity ofany symptoms of PRRSV infection and/or any gross or histopathologicalchange when compared to the results of challenging a non-vaccinated(untreated with the vaccine) pig with the same isolate. Of course thepig should be PRRSV-free, such as a pig that has not been previouslyexposed to the virus or which has been exposed but symptom-free for asufficient period of time to identify it as uninfected. Alternatively,the pig may be identified as uninfected by use of an assay to detect thepresence of PRRSV or anti-PRRSV antibody in a bodily fluid or tissuesample from the pig.

Non-limiting examples of symptoms of PRRSV infection include fever,respiratory distress, cyanosis, pneumonia, lethargy, sneezing, coughing,eye edema, blue ears, and heart and/or brain lesions. Additionally, thepresence of the isolate used in the challenge may be determined by otherquantitative or qualitative methods. Non-limiting examples includedetection of lung lesions, or virus in a blood or serum sample, in achallenged pig, with or without vaccination, after about 2 days to about2 weeks. A decrease in lesions in a vaccinated pig, in comparison to anuntreated pig, provides a quantitative means to detect infection.Alternatively, detection of virus in the blood or serum of a vaccinatedpig indicates that the vaccination may not have been effective while anegative detection of virus indicates that the vaccination may have beeneffective.

The effective stimulation of immunoprotection in a subject may bemediated by the generation of an antibody and/or immune response afterexposure to a combination or composition of the disclosure. Non-limitingexamples of the subject may be a pig that has not been previouslyexposed to PRRSV or a pig that has been exposed to PRRSV or sufferingthe effects from PRRSV infection. In many embodiments, the production ofan antibody response includes the production of neutralizing antibodiesagainst the GP5 protein, including all or part of the HV2 therein.Confirmation of the generation of such antibodies may be performed byassaying blood or serum from a treated animal for the presence of suchantibodies. Non-limiting examples of such antibody detection assaysinclude ELISA, RIA, and Western blotting.

The disclosure includes a method of producing an antibody and/or immuneresponse in a subject as described herein. In some embodiments, themethod comprises at least i) identifying or selecting a first PRRSVisolate comprising a polypeptide molecule containing a first HV-2hypervariable region; ii) identifying or selecting a second PRRSVisolate comprising a polypeptide molecule containing a second HV-2hypervariable region different from said first hypervariable region; andiii) administering the first and second isolates to a subject to producean antibody and/or immune response in said subject. In some embodiments,the method may include selection of one or more additional isolates withadditional different HV-2 regions followed by their administration withthe first and second isolates. In other embodiments, the method mayinclude administration of one or more unselected isolates with the firstand second isolates.

The amount of the first and second isolates to administer should ofcourse be sufficient to produce a desired antibody and/or immuneresponse. In some embodiments, the administered amount is sufficient toproduce a vaccinated or protected state in the subject againstsubsequent PRRSV infection by one or more isolates.

In some cases, the identifying or selecting may comprise i) amino acidsequence analysis of the PRRSV GP5 ectodomain HV-2 hypervariable region;ii) PCR-based or antibody-based detection of the PRRSV GP5 ectodomainHV-2 hypervariable region; or iii) knowing the PRRSV GP5 ectodomain HV-2hypervariable region sequence relative to another isolate.

In other embodiments, a method of producing an antibody and/or immuneresponse comprises administration of a first polypeptide (antigenic)domain comprising an HV-2 region selected from D-1, D-2, D-3, D-4, D-5,D-6, D-7, or D-8, and a second polypeptide (antigenic) domain comprisingan HV-2 region selected from S-1, S-2, S-3, S-4, S-5, S-6, S-7, or S-8.In many cases, the combination with two or more different polypeptides(antigenic) domains selected from D-1, D-2, D-3, D-4, D-5, D-6, D-7,D-8, S-1, S-2, S-3, S-4, S-5, S-6, S-7 and S-8 is advantageously used inNorth America.

In other embodiments, the administration comprises two or morepolypeptide (antigenic) domains selected from E-1, E-2, E-3, E-4, E-5,E-6, E-7, and E-8. In many cases, this combination is advantageouslyused in Europe. In additional embodiments, a combination of two or moredifferent polypeptide (antigenic) domains may be selected from the 24subgroups depending on the PRRSV isolates found in a particulargeographic regions. Non-limiting examples include the isolates found inSouth Korea, China, Japan, Southeast Asia, or South America. Inadditional embodiments, a combination used in S. Korea, China, or Japanmay be the same as one used in North America. In other embodiments, adomain of any of E-1, E-2, E-3, E-4, E-5, E-6, E-7, and E-8 may beexcluded from a combination of domains for use in North America or anAsian location.

In further embodiments, a combination of two or more polypeptide(antigenic) domains, or polypeptide molecules or isolates containingthem, comprising at least one from each of subgroups detected at ageographic region may be administered in the practice of the disclosure.Administration of the polypeptide (antigenic) domains, or polypeptidemolecules or isolates containing them, may be by any suitable meansknown to the skilled person. Non-limiting examples include injection,intranasal administration, or oral administration, of one or moredisclosed isolates or of one or more sample of cells and/or tissue froma PRRSV infected subject.

If administered or applied separately, the domains, or polypeptidemolecules or isolates containing them, may be sequentially administered,with an optional time interval between administrations. Non-limitingexamples of the time interval include about 1 to about 2 days; about 1,about 3, or about 5 weeks; about 1, about 3, about 4 or about 6 months,or longer. The same time intervals may be used in between a primaryadministration event and one or more subsequent “booster” events.

Whether administered together or separately, the polypeptide molecule(s)may be membrane bound or membrane associated, such by association with alipid bilayer. In some cases, the membrane is from a cell, such as afragment of a cellular membrane. In other embodiments, the membrane isthat of a vesicle, such as a liposome, oil-in-water or water-in oilsuspension. Non-limiting examples of a cell derived membrane include theouter membrane of a PRRSV particle or other viral particle as describedherein.

Kits

The polypeptide domains, polypeptide molecules, and isolates, as well ascombinations and compositions comprising them and their methods of usemay be embodied in one or more kits produced in accordance with wellknown procedures. The disclosure thus includes a kit with one or morereagents comprising one or more polypeptide domains, polypeptidemolecules, or isolates, as described herein, or a combination orcomposition comprising them, for use in one or more methods as disclosedherein. Such a kit optionally further comprises an identifyingdescription or label or instructions relating to their its use in one ormore method of the present disclosure. Such a kit may comprisecontainers, each with one or more of the various reagents (typically inconcentrated form) utilized in the methods. A set of instructions willalso typically be included.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present disclosure, unless specified.

EXAMPLES Example 1 Failure of Conserved GP5 Sequence to GenerateProtected State

The disclosure is based in part upon the recognition that pigspreviously infected with a first isolate of PRRSV can recover but besusceptible to a second isolate where both isolates contain a GP5protein with the same sequence in the Conserved Region. Non-limitingexamples of such incidents are shown in Table 2 below, where eachincident involved pigs that recovered from infection with one of theidentified isolates (the first of each incident set in the table) werethen found to be infected with at least one other isolate (the second ofeach of incidents 1-3 and 5-7) as indicated. A portion of the GP5sequence including the ectodomain in each of the isolates is indicated,with the Conserved Region as identified in FIG. 1 underlined anddifferences in the HV2 region indicated in bold.

TABLE 2 Incident/ Relative position number in GP5 protein Isolates21         31         41         51         61 #1 Q-05-30318 VPFCFVALVN ASNNSSSHLQ LIYNLTICEL NGTDWLNKSF D (Group D-7;Seq ID No: 1765) Q-06-15248 VPFCFVALVN ASNNSSSHLQ LIYNLTICEL NGTDWLNKNF D (Group D-6;Seq ID No: 1766) #2 I-03-28077 VPFCFVALVN ASNNSSSHLQ LIYNLTICEL NGTDWLNKSF D (Group D-7;  Seq ID No: 1767) I-04-32332 VPFCFVALVN ASNNSSSHLQ LIYNLTICEL NGTDWLDKTF D (Group D-7;Seq ID No: 1768) #3 S-06-20709 VPFCLAALVN ADSNSSSHLQ LIYNLTICEL NGTDWLNNHF S (Group S-1;Seq ID No: 1769) S-06-20720 VPFCLAALVN ADSNSSSHLQ LIYNLTICEL NGTDWLNNRF G (Group D-5;Seq ID No: 1770) #4 M-05-2912 VPFCFAVLAN ASNNSSSHLQ LIYNLTLCEL NGTDWLANKF D (Group D-1;Seq ID No: 1771) M-06-13702 VPFCLVALVN ANSNNSSHLQ LIYNLTICEL NGTDWLNRHF S (Group S-5;Seq ID No: 1772) M-06-18282 VPFCLVALVN ANSNNSSHLQ LIYNLTICEL NGTDWLNEHF S (Group S-2;Seq ID No: 1773) #5 H-04-10314 VPFCFAALVN ASNNSSSHLQ LIYNLTICEL NGTDWLNEHF S (Group S-2;Seq ID No: 1774) H-06-14421 VPFCFVALVN ASNNSSSHLQ LIYNLTICEL NGTDWLNKNF D (Group D-6;Seq ID No: 1775) #6 A-00-19757 VPFXFAVIVN ANNNSSSHFQ LIYNLTLCEL NGTEWLNKKF D (Group D-4;Seq ID No: 1776) A-00-53953 VPFWFAVLVD ANSNSSSHFQ LIYNLTICEL NGTDWLNNKF D (Group D-5;Seq ID No: 1777) #7 G-00-3628 VPSCFVAPVN ANDNNSSKLQ LIYNLTLCEL NGTDWLAGKF D (Group D-3;Seq ID No: 1778) G-05-6157 VPFCFAVIVN ASNNSSSHFQ LIYNLTLCEL NGTDWLAEHF N (Group D-1;Seq ID No: 1779)

Based upon a study of such incidents, a discovery was made thatantibodies directed against the Conserved Region in a GP5 protein of anisolate are insufficient to provide protection against a subsequentPRRSV. Additionally, the sequences of the HV-1 region did not provide anadequate explanation for the incidents. A majority of the incidentsshown include no change in the HV1 sequence. This led to the discoverythat the sequence variation in the HV-2 region participates in evadingthe immune surveillance of an animal previously exposed to a PRRSV witha different sequence in the HV-2 region. Stated differently, theconserved sequence in the GP5 ectodomain as shown above is unable toproduce an antibody or immune response that is protective againstanother PRRSV with a different HV-2 region in the GP5 protein.

This discovery led, in part, to the disclosed combinations,compositions, and methods.

Example 2 Propagation of PRRSV Isolates

Methods for the propagation and maintenance of PRRSV isolates has beenpreviously reported (see for example Meng et al., 1994, J. Gen. Virol.75:1795-1801 and Meng et al., 1996, J. of Vet. Diag. Invest. 8:374-381).Non-limiting examples include the use of cell line ATCC CRL 11171, whichcan be grown in monolayers suitable for inoculation with a viralisolate. Alternative cells and cell lines include MA-104, PSP-36,PSP-36-SAH, MARC-145 and porcine alveolar macrophages.

As a non-limiting example, a multiplicity of infection (moi) of about0.1, 0.5, or 1 may be used followed by incubation for about 48 hoursprior to confirmation of infection and viral replication. Confirmationmay be by removal of supernatant (culture media) and fixing the cellsfollowed by detection with a labeled anti-PRRSV antibody, such as amonoclonal antibody specific for the N protein (encoded by ORF 7) or anantibody against a particular HV2 region of a GP5 protein as describedherein.

Example 3 Virus Isolate Combinations

As described herein, PRRSV isolates may be classified (identified) andselected for use in a combination of the disclosure at least on thebasis of the HV2 sequence. The following data shows a portion of the GP5sequence (including the ectodomain) in each of numerous representativePRRSV isolates, some of which differ in regions outside the ectodomain.The locations of the HV1, conserved region (CR), and HV2 as describedherein are indicated at the bottom of the data, with the indication ofthe start of the HV1 being a non-limiting representative example.

The classification of the sequences into the disclosed Groups isincluded, and combinations of isolates from different subgroups may beused in the practice of the disclosure. So as one non-limiting example,a combination of a D-4 isolate (Ingelvac-ATP), a D-1 isolate(Ingelvac-MLV or one of MJ-3 to MJ-14), an S-1 isolate (MJ-1 or MJ-2),and a D-3 isolate (MJ-15 or MJ-16) may be used to produce an immuneresponse in a subject as disclosed herein.

Another non-limiting example is a combination of a D-1 isolate (one ofMJ-17 to MJ-27), a D-6 isolate (one of MJ-28 to MJ-30), a D-2 isolate(MJ-34 or MJ-35), and a D-3 isolate (such as MJ-36). All othercombinations of isolates represented by the data below, and inaccordance with the disclosure, are specifically contemplated forpreparation and use as described herein.

All Strains including European Strain (LV) Ingelvac-ATPLVNANSNSSSHLQLIYNLTLCELNGTDWLKDKFD (Group D-4; Seq ID No: 188) VR-2332LANASNDSSSHLQLIYNLTLCELNGTDWLANKFD (Group D-1; Seq ID No: 47)Ingelvac-MLV LANASNDSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 47) Prime-Pac LVNASYSSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 48) MJ-1 LANASSNSSSHLQLIYNLTICELNGTDWLNNHFS(Group S-1; Seq ID No: 1780) MJ-2 LANANSNSSSHLQLIYNLTICELNGTDWLNNHFS(Group S-1; Seq ID No: 1781) MJ-3 LANASNDSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 47) MJ-4 LANASNGSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 75) MJ-5 LANASNHSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1142) MJ-6 LANASNNSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 055) MJ-7 LASASNSSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1782) MJ-8 LATPSPSSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1783) MJ-9 LANASNANSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1784) MJ-11 LANASNVNSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1785) MJ-12 LANASNDNSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1786) MJ-13 LANASNSNSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1787) MJ-14 LANASNSNSSHLQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 1788) MJ-15 LANASNGNSSHLQLIYNLTLCELNGTDWLAGKFD(Group D-3; Seq ID No: 1789) MJ-16 LANASNSSNSHLQLIYNLTLCELNGTDWLAGKFD(Group D-3; Seq ID No: 1790) MJ-17 LANASNDSSSHLQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 1791) MJ-18 LANASNTSSSHLQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 1792) MJ-19 LANASNNSSSHLQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 484) MJ-20 LANANNTSSSHLQLIYNLTLCELNGTDXLAEKFD(Group D-1; Seq ID No: 1793) MJ-21 LANANNSSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1794) MJ-22 LANASNNSSSHLQLIYNLTLCELNGTDWLANQFD(Group D-6; Seq ID No: 1337) MJ-23 LANASSNSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1795) MJ-24 LANASANSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 61) MJ-25 LANASHNSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 508) MJ-26 LANASQNSSSHLQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1796) MJ-27 LANASSNSSSHLQLIYNLTLCELNGTDWLANRFD(Group D-1; Seq ID No: 1797) MJ-28 LANASSDNSSHLQLIYNLTLCELNGTDWLANNFD(Group D-6; Seq ID No: 1798) MJ-29 LANASSDNSSHLQLIYNLTLCELNGTDWLANNFD(Group D-6; Seq ID No: 1798) MJ-30 LASANSINSPHLQLIYNLTLCELNGTDWLAGEFD(Group D-6; Seq ID No: 1799) MJ-31 LASASNNSSSRLQLIYNLTLCELNGTDWLADRFN(Group D-1; Seq ID No: 1800) MJ-32 LADAHSSSSSHLQLIYNLTLCELNGTDWLADRFD(Group D-1; Seq ID No: 1801) MJ-33 LANAGNNSSSHLQLIYNLTLCELNGTEWLAERFD(Group D-1; Seq ID No: 1802) MJ-34 LGSASSNSSSHFQLIYNLTLCELNGTDWLASRFD(Group D-2; Seq ID No: 1803) MJ-35 LVDANNSSSSHFQLIYNLTICELNGTDWLKARFD(Group D-2; Seq ID No: 1804) MJ-36 LVDANNSSSSHFQLIYNLTICELNGTDWLAARFD(Group D-3; Seq ID No: 1805) MJ-37 LVDANGNSSSHLQLIYNLTLCELNGTDWLANRFD(Group D-1; Seq ID No: 1806) MJ-38 LVNANSTSSSHIQLIYNLTLCELNGTDWLGDKFD(Group D-1; Seq ID No: 1807) MJ-39 LVNANSSSSSHIQLIYNLTLCELNGTDWLTNKFD(Group D-4; Seq ID No: 1453) MJ-40 LVNANSSSSSHLQSIYNLTLCELNGTDWLGNKFD(Group D-1; Seq ID No: 1808) MJ-41 LVDANSSSSSHFQLIYNLTLCELNGTDWLNDKFD(Group D-5; Seq ID No: 1809) MJ-42 LVDANSSSSSHFQLIYNLTLCELNGTDWLNEKFD(Group D-5; Seq ID No: 1810) MJ-43 LVNANSSSSSHFQLIYNLTLCELNGTDWLNEKFD(Group D-5; Seq ID No: 1811) MJ-44 LVNANSSSSSHFQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 44) MJ-45 LVNANSSSSSHFQLIYNLTLCELNGTDWLGNKFD(Group D-1; Seq ID No: 464) MJ-46 LANANSSSSSHFQLIYNLTLCELNGTDWLDKKFD(Group D-4; Seq ID No: 1812) MJ-47 LVNANSASSSHSQLIYNLTLCELNGTDWLDGKFE(Group D-1; Seq ID No: 1813) MJ-48 LVNANSASSSHSQLIYNLTLCELNGTDWLAGKFE(Group D-3; Seq ID No: 1814) MJ-49 LVNANSTSSSPFQLIYNLTLCELNGTDWLQGKFN(Group D-1; Seq ID No: 1815) MJ-50 IANASSNSSSHIQLIYNLTLCELNGTDWLAGKFD(Group D-3; Seq ID No: 1816) MJ-51 IVNANSNSSSHIQLIYNLTLCELNGTDWLADKFD(Group D-1; Seq ID No: 952) MJ-52 IVNANSNSSSHFQLIYNLTLCELNGTDWLANKFD(Group D-1; Seq ID No: 1817) MJ-53 VVNANSNSSSHFQSIYNLTLCELNGTKWLATKFD(Group D-2; Seq ID No: 1818) MJ-54 LDNANSTSSSHFQSIYNLTLCELNGTEWLAENFD(Group D-6; Seq ID No: 1819) MJ-55 LDNANSTSSSHFQSIYNLTLCELNGTKWLAEHFD(Group D-1; Seq ID No: 1820) MJ-56 LVNANSTSSSHFQSIYNLTLCELNGTDWLKEKFD(Group D-4; Seq ID No: 1821) MJ-57 LVDANSSSSSHFQSIYNLTLCELNGTDWLTERFD(Group D-4; Seq ID No: 1822) MJ-58 LVNANSNSSSHFQLIYNLTLCELNGTDWLAQKFD(Group D-1; Seq ID No: 1757) MJ-59 LVNANSNSSSHFQLIYNLTLCELNGTDWLAKKFD(Group D-2; Seq ID No: 572) MJ-60 LVDANSNSSSHFQLIYNLTLCELNGPDWLKKNFD(Group D-6; Seq ID No: 1823) MJ-61 LVNANSNSSSHFQLIYNLTLCELNGTDWLKEKFD(Group D-4; Seq ID No: 571) MJ-62 LVGANGNSSSHFQLIYNLTLCELNGTDWLDEKFD(Group D-5; Seq ID No: 1824) MJ-63 LVNASSNSSSHFQLIYNLTLCELNGTDWLKNKFD(Group D-4; Seq ID No: 1825) MJ-64 LVNAHSNSSSHFQSIYNLTLCELNGTDWLDKKFD(Group D-4; Seq ID No: 1826) MJ-65 LVNAHDNSSSHFQLIYNLTLCELNGTDWLNKKFD(Group D-4; Seq ID No: 1827) MJ-66 LVNASNTSSSYFQSIYNLTLCELNGTDWLKDKFD(Group D-4; Seq ID No: 1828) MJ-67 LVNASNSSSSHFQLIYNLTLCELNGTDWLQGKFD(Group D-1; Seq ID No: 1829) MJ-68 IVNASNSNSSHLQSIYSLTLCELNGTEWLGKNFD(Group D-6; Seq ID No: 1830) MJ-69 LVNANNSSSSHFQSIYNLTLCELNGTEWLAKNFN(Group D-6; Seq ID No: 1831) MJ-70 LVNASSNNSSHFQLIYNLTLCELNGTEWLAKNFI(Group D-6; Seq ID No: 1832) MJ-71 LVNANSSSSSHLQLIYNLTLCELNGTDWLKDKFD(Group D-4; Seq ID No: 473) MJ-72 LVNANSNSSSHLQLIYNLTLCELNGADWLKDKFA(Group D-4; Seq ID No: 1833) MJ-73 LVNASNSNSSHLQLIYNLTLCELNGTDWLGNKFN(Group D-1; Seq ID No: 1834) MJ-74 LVNANSNNSSHLQLIYNLTLCSLNGTDWLANKFD(Group D-1; Seq ID No: 1365) MJ-75 LASANNNHSSHLQSIYNLTLCELNGTDWLSDKFD(Group D-4; Seq ID No: 1835) MJ-76 LASANGNHSSHLQSIYNLTLCELNGTDWLRSRFS(Group S-4; Seq ID No: 1836) MJ-77 LVGASNTSSSHFQLIYNLTLCELNGTDWLNNHFY(Group S-7; Seq ID No: 1837) MJ-78 IVDANSNSSSHFQLIYNLTLCELNGTDWLNNHFN(Group D-5; Seq ID No: 1838) MJ-79 LVDANSNSSSHFQLIYNLTLCELNGTDWLNNHFT(Group S-7; Seq ID No: 804) MJ-80 PVNANNGSSSYSQLIYNLTICELNGTDWLNSKFD(Group D-4; Seq ID No: 1839) MJ-81 PVNANNGTSSYSQLIYNLTICELNGTEWLGSKFD(Group D-2; Seq ID No: 1840) MJ-82 LVNAANTSSSYSQLIYNLTLCELNGTDWLVNRFD(Group D-1; Seq ID No: 1841) MJ-83 LANANNTSSSYSQLIYNLTLCELNGTDWLVGKFE(Group D-3; Seq ID No: 1842) MJ-84 LANANSTSSSYSQLIYNLTICELNGTDWLDDNFD(Group D-6; Seq ID No: 1843) MJ-85 LVNANSSSSSYSQLIYNLTLCELNGTDWLDKKFY(Group S-7; Seq ID No: 1844) MJ-86 LVNANNTSSSYSQLIYNLTLCELNGADWLKEHFS(Group S-5; Seq ID No: 1845) MJ-87 LVNANNTNSSYSQLIYNLTLCELNGTDWLKGHFS(Group S-4; Seq ID No: 1846) MJ-88 LVNANSTSSSYSQLIYNLTLCELNGTEWLGNSFN(Group D-7; Seq ID No: 1847) MJ-89 LVNANSTSSSYSQLIYNLTLCELNGTEWLGTKFS(Group S-4; Seq ID No: 1848) MJ-90 LVNANSTSSSYSQLIYNLTLCELNGTEWLGEKFS(Group S-3; Seq ID No: 1849) MJ-91 LVNANSTNSSYSQLIYNLTLCELNGTEWLGKNFS(Group S-8; Seq ID No: 1850) MJ-92 LVNANSTNSSYSQLIYKLTLCELNGTEWLGKKFS(Group S-4; Seq ID No: 1851) MJ-93 LVNANSTSSSYSQLIYNLTLCELNGTDWLNEKFS(Group S-2; Seq ID No: 1852) MJ-94 LVNANSTSSSYSQLIYNLTLCELNGTDWLNDKFS(Group S-2; Seq ID No: 1853) MJ-95 LVNANSTSSSYSQLIYNLTLCELNGTDWLDGHFS(Group S-3; Seq ID No: 1854) MJ-96 LVNANSTSSSYSQLIYNLTICELNGTDWLNGQFS(Group S-3; Seq ID No: 1855) MJ-97 LVNANNTSSSYSQLIYNLTICELNGTDWLNGRFS(Group S-8; Seq ID No: 1856) MJ-98 LVNANNTSSSYSQLIYNLTICELNGTDWLNGKFS(Group S-3; Seq ID No: 1857) MJ-99 LVNANSTSSSYSQLIYNLTICELNGTDWLNEHFS(Group S-2; Seq ID No: 1858) MJ-100 LVNASNNSSSYSQLIYNLTLCELNGTDWLNKKFS(Group S-5; Seq ID No: 1859) MJ-101 LVNASNNSSSHLQLIYNLTICELNGTDWLDKTFD(Group D-7; Seq ID No: 1860) MJ-102 LVNASNNSSSHLQLIYNLTICELNGTDWLDKSFD(Group D-7; Seq ID No: 1861) MJ-103 LVNASNNSSSHLQLIYNLTICELNGTDWLNKTFD(Group D-7; Seq ID No: 1862) MJ-104 LVNASNNSSSHLQLIYNLTICELNGTDWLNKSFD(Group D-7; Seq ID No: 878) MJ-105 LVNASNNSSSHLQLIYNLTICELNGTDWLNRSFD(Group D-7; Seq ID No: 1863) MJ-106 LVNASNNSSSHLQLIYNLTICELNGTDWLNESFD(Group D-7; Seq ID No: 1864) MJ-107 LVNASNNSSSHLQLIYNLTICELNGTDWLSNNFD(Group D-6; Seq ID No: 1865) MJ-108 LVNASNNGSSHLQLIYNLTICELNGTDWLNNTFD(Group D-7; Seq ID No: 1866) MJ-109 LVNANSNSSSHLQLIYNLTICELNGTDWLNDHFS(Group S-2; Seq ID No: 1668) MJ-110 LVNANSNSSSHLQLIYNLTICELNGTDWLNEHFS(Group S-2; Seq ID No: 768) MJ-111 LVNANSNSSSHLQLIYNLTICELNGTDWLNSHFS(Group S-5; Seq ID No: 461) MJ-112 LVNAHSNSSSHLQLIYNLTICELNGTDWLNKHFS(Group S-5; Seq ID No: 949) MJ-113 LVNANSSNSSHLQLIYNLTICELNGTDWLNNHFS(Group S-1; Seq ID No: 1073) MJ-114 LVNASNDSSSHLQLIYNLTICELNGTDWLNGHFS(Group S-3; Seq ID No: 1867) MJ-115 LVNASNSSSSNLQLIYNLTICELNGTDWLKNHFS(Group S-5; Seq ID No: 1868) MJ-116 LVNASSNSSSHLQLIYNLTICELNGTDWLENHFS(Group S-1; Seq ID No: 939) MJ-117 LVNANSNSSSHLQLIYNLTICELNGTDWLKNHFS(Group S-5; Seq ID No: 1022) MJ-118 LVNANSNSSSNLQLIYNLTICELNGTEWLGSHFS(Group S-4; Seq ID No: 1869) MJ-119 LVNADSNSSSHLQLIYNLTICELNGTDWLNNHFG(Group D-5; Seq ID No: 837) MJ-120 LVNANNSSSSHTQLIYNLTLCELNGTEWLSHKFD(Group D-8; Seq ID No: 1870) MJ-121 LVNAANSSSSHFQSIYNLTLCELNGTDWLSKKFD(Group D-8; Seq ID No: 1871) MJ-122 LVNANNTSSSHFQLIYNLTLCELNGTDWLKYKFE(Group D-8; Seq ID No: 1872) MJ-123 LVDANSNSSSHFQLIYNLTICELNGTDWLYKHFD(Group D-8; Seq ID No: 1873) MJ-124 FADGNGNNSTY-QYIYNLTICELNGTNWLSGHFE(Group E-1; Seq ID No: 1874) MJ-125 FADGNGNNSTY-QYIYNLTICELNGTNWLSDHFE(Group E-1; Seq ID No: 1875) MJ-126 FADGNDNNSTY-QYIYNLTICELNGTNWLSAHFE(Group E-1; Seq ID No: 1876) MJ-127 FADGNGNNSTY-QYIYNLTICELNGTDWLSAHFE(Group E-2; Seq ID No: 1877) MJ-128 FADGNGNDSTY-QYIYDLTICELNGTHWLSNHFV(Group E-8; Seq ID No: 1878) MJ-129 FADGNGNDSTY-QYIYNLTICELNGTSWLSDHFE(Group E-4; Seq ID No: 1879) MJ-130 FADGSGNNSTY-QYIYNLTICELNGTDWLSGHFN(Group E-2; Seq ID No: 1880) MJ-131 FADGSGNNSTY-QYIYNLTICELNGTKWLSGHFD(Group E-3; Seq ID No: 1881) MJ-132 FADGNGNSSTY-QYIYNLTICELNGTTWLSGHFN(Group E-4; Seq ID No: 1882) MJ-133 FADGNGNSSTY-QYIYNLTICELNGTNWLSGHFN(Group E-1; Seq ID No: 1883) MJ-134 FADGNGNNSTY-QYIYNLTICELNGTDWLSNHFS(Group E-6; Seq ID No: 1884) MJ-135 FADGNDNNSTY-QYIYNLTICELNGTNWLSNHFS(Group E-5; Seq ID No: 1885) MJ-136 FADGNGDSSTY-QYIYNLTICELNGTDWLSSHFG(Group E-7; Seq ID No: 1886) LV FADGNGDSSTY-QYIYNLTICELNGTDWLSSHFG(Group E-7; Seq ID No: 1887) |←    HV1   →|←C.Region→ |←  HV2→ |

All references cited herein are hereby incorporated by reference intheir entireties, whether previously specifically incorporated or not.As used herein, the terms “a”, “an”, and “any” are each intended toinclude both the singular and plural forms.

Having now fully described the invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the disclosure and without undueexperimentation. While the invention has been described in connectionwith specific embodiments thereof, it will be understood that it iscapable of further modifications. This application is intended to coverany variations, uses, or adaptations of the disclosure following, ingeneral, the principles of the disclosure and including such departuresfrom the present disclosure as come within known or customary practicewithin the art to which the disclosure pertains and as may be applied tothe essential features hereinbefore set forth.

1. A method of classifying a PRRSV isolate, the method comprisingsequencing the HV-2 hypervariable region of a PRRSV isolate, andidentifying or classifying the PRRSV isolate based upon the HV-2hypervariable region.
 2. The method of claim 1, wherein the HV-2hypervariable region is of about eight amino acid residues that follow aconserved GP5 motif, represented by the sequence C(E/S)LNG(T/A), SEQ IDNO:1.
 3. The method of claim 1, wherein the HV-2 hypervariable region isrepresented by the sequence X₀WLX₁X₂X₃X₄X₅, wherein each of X₀, X₁, X₂,X₃, X₄, and X₅ is independently one of the 20 naturally occurring aminoacid residues, except that X₅ is not cysteine, methionine, proline,phenylalanine, or tryptophan.
 4. The method of claim 3, wherein the HV-2region comprises the sequence X₀WLX₁X₂X₃X₄X₅ wherein X₅ is not cysteine,methionine, proline, phenylalanine, tryptophan, serine, threonine, ortyrosine, and is identified or classified as D-1: wherein X₁ is analiphatic amino acid residue, X₂ is an acidic amino acid residue (orwherein X₁ is an acidic amino acid residue and X₂ is an aliphatic aminoacid residue), X₃ is a basic amino acid residue, and X₄ is an amino acidresidue comprising an aromatic ring, such as phenylalanine (F); D-2:wherein X₁ is an aliphatic amino acid residue, X₂ is serine (S),threonine (T), tyrosine (Y) or an basic amino acid residue (or whereinX₁ is serine (S), threonine (T), tyrosine (Y) or a basic amino acidresidue and X₂ is an aliphatic amino acid residue), X₃ is a basic aminoacid residue, and X₄ is an amino acid residue comprising an aromaticring, such as phenylalanine (F); D-3: wherein each of X₁ and X₂ isindependently an aliphatic amino acid residue, X₃ is a basic amino acidresidue, and X₄ is an amino acid residue comprising an aromatic ring,such as phenylalanine (F); D-4: wherein X₁ is an acidic amino acidresidue, X₂ is serine (S), threonine (T), tyrosine (Y) or a basic aminoacid residue (or wherein X₁ is serine (S), threonine (T), tyrosine (Y)or a basic amino acid residue and X₂ is an acidic amino acid residue),X₃ is a basic amino acid residue, and X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F); D-5: whereineach of X₁ and X₂ is independently an acidic amino acid residue, X₃ is abasic amino acid residue, and X₄ is an amino acid residue comprising anaromatic ring, such as phenylalanine (F); D-6: wherein each of X₁ and X₂is independently one of the 20 naturally occurring amino acid residues,X₃ is an acidic amino acid residue, and X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F); D-7: whereineach of X₁ and X₂ is independently one of the 20 naturally occurringamino acid residues, X₃ is a non-aromatic amino acid residue with ahydroxyl containing R-group, and X₄ is an amino acid residue comprisingan aromatic ring, such as phenylalanine (F); or D-8: wherein each of X₁and X₂ is independently serine (S), threonine (T), tyrosine (Y) or abasic amino acid residue, X₃ is a basic amino acid residue, and X₄ is anamino acid residue comprising an aromatic ring, such as phenylalanine(F).
 5. The method of claim 1, wherein the HV-2 region comprises thesequence X₀WLX₁X₂X₃X₄X₅ and is identified or classified as S-1: whereinX₁ is an acidic amino acid residue, X₂ is asparagine (N), X₃ is a basicamino acid residue, X₄ is an amino acid residue comprising an aromaticring, such as phenylalanine (F), and X₅ is serine (S); S-2: wherein eachof X₁ and X₂ is independently an acidic amino acid residue except thatX₂ is not asparagine (N), X₃ is a basic amino acid residue, X₄ is anamino acid residue comprising an aromatic ring, such as phenylalanine(F), and X₅ is serine (S); S-3: wherein X₁ is an aliphatic amino acidresidue, X₂ is an acidic amino acid residue (or wherein X₁ is an acidicamino acid residue and X₂ is an aliphatic amino acid residue), X₃ is abasic amino acid residue, X₄ is an amino acid residue comprising anaromatic ring, such as phenylalanine (F), and X₅ is serine (S); S-4:wherein X₁ is an aliphatic amino acid residue, X₂ is serine (S),threonine (T), tyrosine (Y) or a basic amino acid residue (or wherein X₁is serine (S), threonine (T), tyrosine (Y) or a basic amino acid residueand X₂ is an aliphatic amino acid residue; or where each of X₁ and X₂ isindependently serine (S), threonine (T), tyrosine (Y) or a basic aminoacid residue; or where each of X₁ and X₂ is independently an aliphaticamino acid residue), X₃ is a basic amino acid residue, X₄ is an aminoacid residue comprising an aromatic ring, such as phenylalanine (F), andX₅ is serine (S); S-5: wherein X₁ is an acidic amino acid residue, X₂ isserine (S), threonine (T), tyrosine (Y) or a basic amino acid residue(or wherein X₁ is serine (S), threonine (T), tyrosine (Y) or a basicamino acid residue and X₂ is an acidic amino acid residue except N(Asn)), X₃ is a basic amino acid residue, X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F), and X₅ is serine(S); S-6: wherein X₁ is serine (S), threonine (T), tyrosine (Y) or abasic amino acid residue, X₂ is an asparagine (N), X₃ is a basic aminoacid residue, X₄ is an amino acid residue comprising an aromatic ring,such as phenylalanine (F), and X₅ is serine (S); S-7: wherein each of X₁and X₂ is independently one of the 20 naturally occurring amino acidresidues, X₃ is a basic amino acid residue, X₄ is an amino acid residuecomprising an aromatic ring, such as phenylalanine (F), and X₅ isthreonine (T) or tyrosine (Y); or S-8: wherein X₁ is an acidic aminoacid residue, X₂ is an acidic amino acid residue (or wherein X₁ is anacidic amino acid residue and, X₂ is an aliphatic amino acid residue, oralternatively wherein X₁ is an aliphatic amino acid residue and, X₂ isan acidic amino acid residue), X₃ is an acidic amino acid residue, X₄ isan amino acid residue comprising an aromatic ring, such as phenylalanine(F), and X₅ is serine (S).
 6. The method of claim 1, wherein the HV-2region comprises the sequence NWLSX₂X₃X₄X₅, wherein each of X₂, X₃, andX₄ is independently one of the 20 naturally occurring amino acidresidues, and X₅ is an acidic amino acid residue, and is identified orclassified as E-1; the sequence X₀WLX₁X₂X₃X₄X₅, wherein X₀ is an acidicamino acid residue except for asparagine (N), each of X₁, X₂, X₃, and X₄is independently one of the 20 naturally occurring amino acid residues,and X₅ is an acidic amino acid residue, and is identified or classifiedas E-2; the sequence X₀WLX₁X₂X₃X₄X₅, wherein X₀ is a basic amino acidresidue, each of X₁, X₂, X₃, and X₄ is independently one of the 20naturally occurring amino acid residues, and X₅ is an acidic amino acidresidue, and is identified or classified as E-3; the sequenceX₀WLX₁X₂X₃X₄X₅, wherein X₀ is any non-acidic and non-basic amino acidresidue, each of X₁, X₂, X₃, and X₄ is independently one of the 20naturally occurring amino acid residues, and X₅ is an acidic amino acidresidue, and is identified or classified as E-4; the sequenceNWLSX₂X₃X₄X₅, wherein each of X₂, X₃ and X₄ is independently one of the20 naturally occurring amino acid residues, and X₅ is Serine (S) orThreonine (T), and is identified or classified as E-5; the sequenceX₀WLX₁NX₃X₄X₅, wherein X₀ is any amino acid residue except asparagine(N), each of X₁, X₃, and X₄ is independently one of the 20 naturallyoccurring amino acid residues, and X₅ is Serine (S) or Threonine (T),and is identified or classified as E-6; the sequence X₀WLX₁X₂X₃X₄X₅,wherein X₀ is an acidic amino acid residue except asparagine (N), eachof X₁, X₂, X₃, and X₄ is independently one of the 20 naturally occurringamino acid residues except that X₂ is not asparagine (N), and X₅ is anynon-acidic amino acid residue, and is identified or classified as E-7;or the sequence X₀WLX₁X₂X₃X₄X₅, wherein X₀ is any non-acidic amino acidresidue, each of X₁, X₂, X₃, and X₄ is independently one of the 20naturally occurring amino acid residues, and X₅ is any non-acidic aminoacid residue, and is identified or classified as E-8.
 7. The method ofclaim 1, wherein the isolate is a PRRSV viral particle.
 8. The method ofclaim 7, wherein the viral particle is a virion.
 9. The method of claim1, wherein the isolate is obtained from a pig.
 10. The method of claim1, wherein the sequencing is of the GP5 coding sequence (ORFS) of theisolate.
 11. The method of claim 1, wherein the sequencing is of the GP5protein.